Prion protein (PrP) underlies a spectrum of neurodegenerative diseases with no established treatment and devastating human and economical consequences. The existence of transmissible forms separates prion diseases from other neurodegenerative illnesses. The "protein only hypothesis" postulates that an abnormal isoform of PrP (PrPSc) acts as an infectious agent and propagates its pathological conformation using normal isoform (PrPC) as a substrate. The ultimate direct proof of the "protein only hypothesis", the reconstitution of PrPSc in vitro from noninfectious PrP, has not been achieved yet. The proposed research aims at reconstitution of the process of prions conversion in vitro following by bioassay in cultured cells and animals. Three strategies are suggested: (i) manipulating conformational properties of recombinant PrP (rPrP) in vitro; (ii) increasing the susceptibility of cells for prion transmission; and (iii) utilizing permanent non protein components of prion plaques to assist in vitro refolding. The use of cultured neuroblastoma cells instead of animals as an intermediate step for transmissible agent allows one to reduce the degree of complexity and to determine factors essential for prion transmission. Several hypotheses proposed by the PI are elaborated into three specific aims: (1) we will determine the capabilities the transient intermediates and end-product of the amyloid formation of rPrP to induces prion propagation in cultured cells; (2) we will explore the capabilities of complexes of a-isoforms of rPrP with non-protein component of prion plaques as transmissible agents; (3) we will examine whether or not glycosylation-inhibiting conditions increases susceptibility of cells for prion replication induced by isoforms of rPrP. Proteinase K digestion, detergent solubility, and immunoconformational assays will be used to detect the formation of nascent PrPSc isoform in cultured cells. Bioassay will be used to validate formation of PrPSc in cultured cells. When rPrP-dependent cellular system of prion propagation is developed, new opportunities will arise for studying detailed mechanisms of transmission and propagation of prions in living cells using non-invasive state-of-the-art biophysical techniques. Such knowledge should lay the foundation for novel molecular and pharmacological approaches of treating transmissible and sporadic prion diseases.